The treatment of human diseases through the administration of nucleic acid-based drugs such as DNA and RNA has the potential to revolutionize the field of medicine (Anderson Nature 392 (Suppl.):25-30, 1996; Friedman Nature Med. 2:144-147, 1996; Crystal Science 270:404-410, 1995; Mulligan Science 260:926-932, 1993; each of which is incorporated herein by reference). Thus far, the use of modified viruses as gene transfer vectors to introduce nucleic acids into cells has generally represented the most clinically successful approach to gene therapy. While viral vectors are currently the most efficient gene transfer agents, concerns surrounding the overall safety of viral vectors, which include the potential for unsolicited immune responses, have resulted in parallel efforts to develop non-viral alternatives (for leading references, see, Luo et al. Nat. Biotechnol. 18:33-37, 2000; Behr Acc. Chem. Res. 26:274-278, 1993; each of which is incorporated herein by reference). Current alternatives to viral vectors include polymeric delivery systems (Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem. 6:7-20, 1995; each of which is incorporated herein by reference), liposomal formulations (Miller Angew. Chem. Int. Ed. 37:1768-1785, 1998; Hope et al. Molecular Membrane Technology 15:1-14, 1998; Deshmukh et al. New J. Chem. 21:113-124, 1997; each of which is incorporated herein by reference), and “naked” DNA injection protocols (Sanford Trends Biotechnol. 6:288-302, 1988; incorporated herein by reference). While these strategies have yet to achieve the clinical effectiveness of viral vectors, the potential safety, processing, and economic benefits offered by these methods (Anderson Nature 392 (Suppl.):25-30, 1996; incorporated herein by reference) have ignited interest in the continued development of non-viral approaches to gene therapy (Boussif et al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; Putnam et al. Macromolecules 32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999; Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999; Kukowska-Latallo et al. Proc. Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al. Bioconjugate Chem. 7:703-714, 1996; Haensler et al. Bioconjugate Chem. 4:372-379, 1993; each of which is incorporated herein by reference).
One form of gene therapy, genetic vaccination, has tremendous potential for treating or preventing numerous diseases for which traditional vaccination has been shown to be effective. Genetic vaccination also may prove effective in treating and preventing diseases for which traditional vaccines are ineffective (Gurunathan et al. DNA vaccines: Immunology, application, and optimization. Annu. Rev. Immunol. 18:927-974 (2000); McKenzie et al. Nucleic acid vaccines—Tasks and tactics. Immunol. Res. 24:225-244 (2001); each of which is incorporated herein by reference). However, this potential is largely unrealized due to the inability of current vaccine systems to safely cause an appropriate level of immunogenicity and target gene expression in antigen presenting cells (APC) (Arthur et al. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther. 4:17-25 (1997); Dubensky, T. W., Jr., Liu, M. A. & Ulmer, J. B. Delivery systems for gene-based vaccines. Mol Med 6:723-732. (2000); Walter, E. & Merkle, H. P. Microparticle-mediated transfection of non-phagocytic cells in vitro. J. Drug Target. 10:11-21 (2002); Denis-Mize, K. S. et al. Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells. Gene Ther. 7:2105-2112 (2000); each of which is incorporated herein by reference). This deficiency is a particularly important issue in non-viral genetic vaccine cancer therapies in which epitopes can be weakly antigenic, and tumors can down-regulate the ability of APCs to process and present antigen efficiently to T-cells in an activated state (Pardoll, Spinning molecular immunology into successful immunotherapy. Nature Reviews Immunology 2:227-238 (2002); incorporated herein by reference). Viral vectors, such as adenovirus, have been shown to transfect dendritic cells in vitro and elicit strong, antigen-specific immune responses in vivo (Walter, E., Thiele, L. & Merkle, H. P. Gene delivery systems to phagocytic antigen-presenting cells. STP Pharma Sci. 11:45-56 (2001); Casimiro, D. R. et al. Vaccine-induced immunity in baboons by using DNA and replication-incompetent adenovirus type 5 vectors expressing a human immunodeficiency virus type 1 gag gene. J. Virol. 77:7663-7668 (2003); Song, W. et al. Dendritic cells genetically modified with an adenovirus vector encoding the CDNA for a model antigen induce protective and therapeutic antitumor immunity. J. Exp. Med. 186:1247-1256 (1997); Wong, C. P. & Levy, R. Recombinant adenovirus vaccine encoding a chimeric T-cell antigen receptor induces protective immunity against a T-cell lymphoma. Cancer Res. 60:2689-2695 (2000); each of which is incorporated herein by reference); however, as mentioned above, there are concerns related to the safety, manufacturability, immunological rejection, and payload size constraints inherent to viral gene delivery (Wickham, T. J. Targeting adenovirus. Gene Ther. 7:110-114 (2000); Tuettenberg et al. Priming of T cells with ad-transduced DC followed by expansion with peptide-pulsed DC significantly enhances the induction, of tumor-specific CD8(+) T cells: implications for an efficient vaccination strategy. Gene Ther. 10:243-250 (2003); Luo, D. & Saltzman, W. M. Synthetic DNA delivery systems. Nat. Biotechnol. 18:33-37 (2000); Clark et al. Gene delivery of vaccines for infectious disease. Curr Opin Mol Ther 3:375-384. (2001); each of which is incorporated herein by reference). Current non-viral vaccine systems are not designed to activate APCs (McKeever et al. Protective immune responses elicited in mice by immunization with formulations of poly(lactide-co-glycolide) microparticles. Vaccine 20:1524-1531 (2002); incorporated herein by reference), and lack the gene delivery capacity of viral vectors. In an attempt to increase immunogenicity of non-viral systems, focus has shifted towards exploring the use of adjuvants, cytokines, and self-replicating RNA systems (Leitner, W. W. et al. Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat. Med. 9:33-39 (2003); Pachuk, C. J., McCallus, D. E., Weiner, D. B. & Satishchandran, C. DNA vaccines—challenges in delivery. Curr. Opin. Mol. Ther. 2:188-198 (2000); Leitner, W. W., Hammerl, P. & Thalhamer, J. Nucleic acid for the treatment of cancer: Genetic vaccines and DNA adjuvants. Curr. Pharm. Design 7:1641-1667 (2001); O'Hagan, D. T., MacKichan, M. L. & Singh, M. Recent developments in adjuvants for vaccines against infectious diseases. Biomol Eng 18:69-85. (2001); each of which is incorporated herein by reference). The ideal non-viral genetic vaccine delivery system would be virus-like in function (Luo, D. & Saltzman, W. M. Synthetic DNA delivery systems. Nat. Biotechnol. 18:33-37 (2000); incorporated herein by reference), i.e., capable of mediating efficient intracellular delivery of antigen-encoding DNA while enhancing the immogenicity of the delivery system.
A promising method of non-viral delivery for genetic vaccines is microparticulate DNA delivery systems formulated with a biodegradable polymers such as poly lactic-co-glycolic acid (PLGA), as these particles take advantage of size-based immunogenicity and APC targeting (O'Hagan et al. Poly(lactide-co-glycolide) microparticles for the development of single-dose controlled-release vaccines. Adv. Drug Deliv. Rev. 32:225-246 (1998); Hedley, M. L., Curley, J. & Urban, R. Microspheres containing plasmid-encoded antigens elicit cytotoxic T-cell responses. Nat. Med. 4:365-368 (1998); O'Hagan et al. Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines. J. Virol. 75:9037-9043 (2001); each of which is incorporated herein by reference). Despite these advantages, even low molecular weight PLGA systems need two weeks to fully release their encapsulated payloads after dendritic cell uptake in vitro (Walter et al. Hydrophilic poly(DL-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells. J. Control. Release 76:149-168 (2001); incorporated herein by reference). This is an excessively long period of time given new results which suggest that most dendritic cells die 7 days after external stimulus and migration to the lymph nodes. Furthermore, PLGA microparticles can produce an extremely low pH microclimate (pH<2 after 3 days in an aqueous environment) (Fu, K., Pack, D. W., Klibanov, A. M. & Langer, R. Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres. Pharm. Res. 17:100-106 (2000); incorporated herein by reference) which reduces the activity of plasmid DNA released from these particles (Walter, E., Moelling, K., Pavlovic, J. & Merkle, H. P. Microencapsulation of DNA using poly(DL-lactide-co-glycolide): stability issues and release characteristics. J. Control. Release 61:361-374 (1999); incorporated herein by reference). PLGA also lacks the ability to facilitate phagosomal escape of the microparticles, and trigger intracellular release. As a result, PLGA microparticles remain confined in phagolysosomal vesicles, resulting in low gene expression (Walter, E., Thiele, L. & Merkle, H. P. Gene delivery systems to phagocytic antigen-presenting cells. STP Pharma Sci. 11:45-56 (2001); incorporated herein by reference).
There exists a continuing need for non-viral immunogenic and non-immunogenic drug delivery systems that allow for the rapid release of their payloads intracellularly, especially in the context of gene therapy with the delivery of fragile biomolecules such as DNA.